Many successful viral vaccines use live attenuated virus strains. The rate-limiting step for live-virus vaccine development is the identification of a suitable attenuated virus. Conventional methods of developing an attenuated virus involve propagation of the virus under novel conditions, such as passage of the virus in “foreign” or non-permissive cell lines, so that it becomes less pathogenic to its original host as it evolves under the new conditions. Although this methodology has shown remarkable success, little is known about the process by which the attenuating mutations arise and evolve, and isolating an attenuated virus is a random, slow process. In addition, the outcome of an attempted attenuation is largely unpredictable, and depending on the nature of the attenuation, an attenuated virus may revert to virulence.
Some currently used attenuation methods result in virus vaccine strains that are too virulent to produce. Highly virulent strains are deleterious to the host and developing vaccines requires inactivation of the virus. This has certain drawbacks. For example, inactivated viruses as orally or nasally applied vaccines must be given in high concentrations in order to bring about a significant increase of antibodies. As another example, the administration of inactivated influenza virus or antigen in convenient commercial doses, free of side effects, with nasal or oral administration, does not produce a satisfactory immune response without the use of an adjuvant. (Chen et al., 1989, Current Topics in Microbiology and Immunology 146:101 106, Couch et al., 1997, J. Infect. Dis. 176:38 44). Thus, for example, for the optimum induction of the immune response with oral administration of an emulsion-inactivated vaccine, an antigen content between 66 μg antigen/dose and 384 μg antigen/dose is required (Avtushenko et al., 1996, J. Biotechnol. 44:21 28). Thus, this dose lies far above that of an inactivated vaccine for parenteral administration, which is at approximately 15 μg antigen/dose.
A cold-adapted, live attenuated influenza virus vaccine to be found in clinical studies for nasal administration is based on virus antigens from which reassortments must be produced annually by means of genetic methods, in which the genes for the hemagglutinin and neuramidase antigens of the corresponding influenza A or B strain are transferred to an attenuated, cold-adapted master virus strain. This method is very time consuming and labor intensive. In addition, there is the danger that through reversion the attenuated virus back mutates into a virulent virus and thus can trigger viremia. When immunization is carried out with living viruses there is also a further spread in the body of the immunized individual. When cold-adapted viruses are used, there is also the constant necessity of storing the virus vaccine below the freezing point, as close to −20° C. as possible, which then requires the absolute maintenance of a chain of refrigeration to ensure sufficient storage life of the vaccine.
Eggs are used for the production of the live attenuated influenza virus vaccine virus reassortments and the propagation of the vaccine viruses, which entails the risk that any contaminating infectious agents that may be present may be transferred into the eggs. The purification of live viruses is also not without problems because they represent infectious material and thus a higher standard of security must be maintained.
The availability of a technique for attenuating viruses in a rapid and non-random way would eliminate the disadvantages associated with current methods.